Molecular docking unveils the potential of andrographolide derivatives against COVID-19: an in silico approach

Background The recent severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection cause high mortality and there is an emergency need to develop a specific drug to treat the novel coronavirus disease, COVID-19. However, some natural and synthetic products with action against SARS-CoV-2 have been reported in recent research, there is no specific drug available for treating COVID-19. In the present study, molecular interaction analysis was performed for 16 semisynthetic andrographolides (AGP) against 5 SARS-CoV-2 enzymes main protease (Mpro, PDB: 6LU7), papain-like protease (PLpro, PDB: 6WUU), spike glycoprotein (S, PDB: 6VXX), NSP15 endoribonuclease (NSP15, PDB: 6VWW), and RNA-dependent RNA polymerase (RdRp, PDB: 6M71). Moreover, the compounds pharmacokinetic and toxic profiles were also analyzed using computational tools. Results The protein−ligand docking score (kcal/mol) revealed that all the tested AGP derivatives showed a better binding affinity towards all the tested enzymes than hydroxychloroquine (HCQ). Meanwhile, all the tested AGP derivatives showed a better binding score with RdRp and S than remdesivir (REM). Interestingly, compounds 12, 14, and 15 showed a better binding affinity towards the all the tested enzyme than AGP, REM, and HCQ. AGP-16 had shown − 8.7 kcal/mol binding/docking score for Mpro, AGP-15 showed − 8.6 kcal/mol for NSP15, and AGP-10, 13, and 15 exhibited − 8.7, − 8.9, and − 8.7 kcal/mol, respectively, for S. Conclusion Overall results of the present study concluded that AGP derivatives 14 and 15 could be the best ‘lead’ candidate for the treatment against SARS-CoV-2 infection. However, molecular dynamic studies and pharmacological screenings are essential to developing AGP derivatives 14 and 15 as a drug against COVID-19. Supplementary Information The online version contains supplementary material available at 10.1186/s43141-022-00339-y.


Background
In the twenty-first century, coronavirus disease−2019 (COVID- 19), one of the most destructive pandemics, was identified after an abnormal pneumonia outbreak in Wuhan, China, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [1]. It generated a global health emergency by its rapid transformation and higher mortality and disrupted the normal human life [2].
The versatility of AGP as a SARS-CoV-2 anti-viral is demonstrated by its potential to bind to several important targets (spike protein-ACE-2 receptor complex, spike protein, ACE-2 receptor, RdRp, 3CLpro, PLpro, and N-protein RNA-binding domain) at various stages of viral attachment, replication, and host-pathogen interactions [9,[11][12][13][14][15][16][17][18][19]. This property may be a vital lead to any potential therapeutic agent being developed. Molecular dynamics study of AGP with SARS-CoV-2 suggests that AGP can bind to both SARS-CoV-2 spike protein and human ACE-2 receptors [11,15]. There was no andrographolide or any compound of A. paniculata in the top 6 potential hits when an in silico and in vitro high throughput screening of 10,000 compounds containing natural compounds was conducted to identify potent 3CLpro inhibitors. Some researchers reported that andrographolide and its natural analogues had less binding affinity than remdesivir or other phytochemicals such as curcumin. However, andrographolide and related compounds were predicted to have binding affinity to several key components of the SARS-CoV-2 life cycle and pathogenicity [9,11,[13][14][15][16][17][18][19]. In addition, andrographolide and the major bioactive compounds of A. paniculata may have additional pleiotropic effects on post-viral infection due to their reported anti-inflammatory and immunomodulatory properties [20][21][22][23][24][25].
Exploring analogues of andrographolide as antivirals may be helpful as these compounds have been suggested to have better binding affinities towards viral targets than the parent andrographolide structure. To date, no specific medications have been developed. Therefore, considering the risk factors associated with this disease, there is an urgent need for a treatment method to treat this disease to limit the transmission [26]. Artificial intelligence plays a crucial role in drug design and discovery to reduce time and cost, mainly used in the repurposing of FDA approved drugs and to find the application of natural, synthetic, and semisynthetic derivatives in the treatment of various diseases, disorders, and infections. Hence, we have done docking studies on some semisynthetic and designed AGP derivatives, with hypothesized that the semisynthetic and designed AGP derivatives would have better interaction affinity towards the main targets of COVID-19 disease than AGP, and remdesivir and hydroxychloroquine, the drugs that are showing better affinity against COVID-19 disease targets. All the reported semisynthetic and a few of our designed AGP derivatives showed better affinity than AGP and used drugs. This study provides some preliminary scientific evidence for the use of AGP derivatives to combat SARS-CoV-2. However, further in vitro and in vivo experimental studies need to be done to confirm the anti-COVID action potential of AGP derivatives.

Preparation of ligands
AGP structures were drawn using the Chem Draw tool, converted to 3D structure using the Chem3D tool in Chem Office-16, and saved as either .sdf or .mol file for further use.

In silico physicochemical and ADME/T studies
Physicochemical, pharmacokinetic and toxicity of AGP derivatives were calculated using online tools Swis-sADME and ProTox-II.

Preparation of proteins
The crystallographic structures of SARS-CoV-2 proteins were checked for broken chain and errors using Swiss Protein Data Base Viewer and corrected. Then, the water and other heteroatoms were removed, polar hydrogens were added to the protein structure, charges assigned, and saved as PDB format for docking study [27].

Docking studies
AGP derivatives and protein structures were uploaded in the Virtual Screening software interface PyRx 0.8 utilizes Autodock Vina and Autodock 4.2. These structures were then energy-minimized using the conjugate gradient optimization algorithm (200 iterations) with MMFF94 forcefield. Local search method Broyden-Fletcher-Goldfarb-Shanno (BFGS) used to energy minimization of protein. Then, the protein and ligand molecules were converted to the '.pdbqt' format using auto dock tools. The active binding site grid box was generated automatically by PyRx. The size and coordinate of grid box were adjusted by entering the calculated values from discovery studio visualizer in the appropriate box [27]. Blind docking was carried out for RNA-dependent RNA polymerase and spike glycoprotein (closed), since the electron microscopic structure of these proteins are available as apoproteins. All other software parameters were kept as default and ligand were considered as flexible and receptor as rigid. PyRx uses Lamarckian genetic algorithm as scoring function. The final visualization of the docked structure was performed using Discovery Studio Visualizer 3.0. The best conformer was selected based on the docking score and better non-covalent bond interaction. The docking pose and interactions pictures were collected and saved [28].

Results
In the present research work, molecular interactions of AGP derivatives with SARS-CoV-2 viral proteins were studied. In this study, we used a total of 17 semisynthetic AGP derivatives ( Fig. 1) with andrographolide, remdesivir, and hydroxychloroquine. In the selected AGP derivatives, nine were taken from the earlier reported study [29] and eight were knowledge based designed compounds. PDB structures of RdRp, PLpro, Mpro, NSP15, and spike protein were given in Fig. 2.

Physicochemical and toxicity properties
The SwissADME web tool was used to compute the physicochemical properties, which was used to identify the drug-likeness of AGP derivatives. The compounds that satisfied Lipinski rule of five for drug-likeness was considered for further study. The physicochemical and pharmacokinetic properties of the AGP derivatives were depicted in Table 1. The AGP derivatives toxicity profile was predicted using online tool ProTox-II, and the toxicity details were tabulated in Table 2.

Active site
The active binding site of the SARS-CoV-2 proteins was predicted using Discovery studio visualizer for the crystallographic structure of protein complex with ligand, and the coordinate/center of the x, y, and z axes of the spheres of each protein active binding site was given in Table 3. A blind docking procedure was adopted for the apoprotein.

Docking study
The present docking study was validated by re-docking extracted ligand from the crystallographic protein-ligand complex structure with the same protein. All binding conformations of the re-docked ligand within the binding pocket of protein produced by the PyRx 0.8 tool were like the binding mode of the co-crystallized ligand, and   the root means square deviation (RMSD) for these conformations were below 2 Å. The docking method for NSP15 (PDB: 6VWW) and RNA-dependent RNA polymerase (PDB: 6M71) was validated by repeated docking with remdesivir. The output was the same for multiple times of docking means the interaction took place in the same site with the identical residues, and the root means square deviation (RMSD) for these conformations were below 2 Å, hence the docking studies for AGP derivatives were carried out using PyRx 0.8 tool. The binding affinity/energy of the AGP derivatives towards SARS-CoV-2 proteins were given in Table 4, and the details of interacting amino acids of proteins with AGP derivatives with the highest binding energy were given in Table 5.
The interacting amino acids of SARS-CoV-2 proteins with all AGP derivatives and reference drugs were given in Table S1 as supplementary data. The PyRx docking output '.pdbqt' files of AGP were analyzed in the discovery studio visualizer. The 3D interaction, docking pose with hydrogen bond donor surface, and 2D interaction image of docking of AGP derivatives on SARS-CoV-2 proteins were generated using a discovery studio visualizer. The docking pose and interaction images of REM, HCQ, and AGP derivatives have the highest binding energy with Mpro, PLpro, RdRp, NSP15, and S were shown in Figs. 3, 4, 5, 6, and 7, respectively.

Binding to Mpro
In-depth molecular docking analysis of AGP derivatives with the active sites of Mpro was performed to understand the protein-ligand interactions. Remdesivir and hydroxychloroquine were used as reference drugs to compare the interaction. Top-ranked conformations were selected based on the binding affinity to the reported binding channel of Mpro by hydrogen bonds and other non-covalent interactions.

Binding to PLPro
Remdesivir and hydroxychloroquine were used as reference drugs to compare the interaction. Top-ranked conformations were selected based on the binding affinity to the reported binding channel of PLpro by hydrogen bonds and other non-covalent interactions. The hydrogen bond interaction surface poses and 2D interactions of REM, HCQ, AGP and AGP-14 on PLpro were shown in Fig. 3A-H. REM is an anti-viral drug that showed the molecular interaction towards the active pocket residue of PLpro with a binding score of − 7.4 kcal/mol. Two H-bonds with Gly163, Asp164, one hydrophobic, two C-H, and one electrostatic interaction (Fig. 4A, B) were noticed in the REM/PLpro complex. While three H-bonds at Ala246, Tyr273, and Asp302, two hydrophobic, one electrostatic and a C-H interaction were observed for HCQ/PLpro complex with a binding score of − 5.7 kcal/mol (Fig. 4C, D).

Discussion
The present study was aimed to identify effective semisynthetic andrographolide derivatives that could interact with various viral proteins and actively reduce or hinder the activity replication of the SARS-CoV-2 virus. Here  contains cysteine and histidine that forms a catalytic dyad, making it an ideal therapeutic target [30]. PLpro is another enzyme responsible for SARS-CoV-2 replication by the action of its catalytic triad (Cys, His, and Asp) and reason for the inflammation of host cells. The viral replication and inflammation of the host cell was reduced by the compounds that bind with catalytic triad [31].
Viruses enter the host cell by binding with host ACE-2 receptor through viral spike protein. The ability of the virus attach to the host cell can be inhibited if spike glycoprotein is inhibited. Endoribonuclease NSP15 is vital for the replication and life cycle of the virus. Hence, we have selected these five enzymes for our current study to explore the interaction efficiency and binding affinity of semisynthetic AGP derivatives on SARS-CoV-2 virus. The drug-likeness and ADMET properties of 17 AGP derivatives and a two FDA approved drugs (remdesivir and hydroxychloroquine, for comparison purposes only) were predicted using SwissADME and ProTox-II online tools, respectively. Lipinski's rule of 5 states that a molecule is considered drug-like when it satisfies: molecular weight < 500 Dalton, number of H-bonds donors < 5, number of H-bonds acceptors < 10 and LogP < 5 [32]. Out of 17 AGP derivatives, except compounds 8, 14, 15, and 16, all other derivatives followed Lipinski's rule of five and were given in Table 1. In that, compounds 8 and 16 has molecular weight > 500, and compounds 14 and 15 has LogP value > 5, but the difference is more negligible. However, the molecular weight of compounds 8 and 16 was marginally lower than REM, and REM also has a high count of hydrogen bond acceptor and rotatable bonds. Hence all the 17 AGP derivatives were considered for docking studies. Only compound 15 showed BBB permeant property like HCQ, and all the AGP derivatives exhibited GI absorption except compounds 6 and 16. Meanwhile, all the 17 AGP derivatives displayed an excellent biological activity score with a value of 0.55, and it is better than the REM value of 0.17 (Table 1). The toxicity prediction showed that all the APG derivatives are free from hepatotoxicity, carcinogenicity, mutagenicity, and cytotoxicity. However, the toxicity prediction results indicated that all the AGP derivatives are immuno-toxic except compound 16. Overall toxicity results indicated that compounds 3, 9, and 17 are safer than other APG derivatives, even though compound 8 showed a better LD 50 value. The acute oral toxicity for all compounds was estimated as class IV except for two compounds, compound 8 in class VI and 11 in class V. The net outcome of toxicity prediction indicated that all 17 AGP derivatives are safer than REM and HCQ (Table 2). Lindner et al. (2005) reported that Mpro or NSP5 is a critical enzyme in forming non-structural proteins. It plays a role in the maturation of other nsp and promotes the biosynthesis of the virus. Mpro can be considered a promising target for the treatment of COVID-19 since there is no known Mpro homologous in humans [33]. The co-crystalized compound N3 in the crystal structure of Mpro showed H-bond interaction with His164, Glu166, Gln189, Thr190, Gly143, Phe140, and hydrophobic interaction with His41, Met49, Met165, Leu167, and Pro168. Significant interactions were observed for all the AGP derivatives used in the study than HCQ with catalytic residues and surface accessible residues of Mpro. However, AGP-10 to 17 had shown a better binding score with Mpro than REM. The four amino acid residues involved in the H-bond formation of AGP-11 with Mpro are the same as the residues involved in the H-bond interaction of N3 and REM, which indicates AGP-11 has a greater number of vital H-bond interaction with Mpro than AGP and AGP-16. Both the cyclic hydroxyl groups of AGP were involved in H-bond formation with Mpro; however, both cyclic hydroxyl groups of AGP-16 were not involved. But AGP-16 showed significant hydrophobic interaction with three residues of Mpro as identical as N3 and REM than AGP-11, which had another amino acid in the interaction. Methyl group and the aromatic ring of sulphanilamide substitution of AGP-16 were involved in the hydrophobic interaction. The present study revealed that the most prominent amino acids that formed interactions with the ligand were Thr24, His41, Met49, Gly143, Met165, Glu166, and Gln189.
These AGP derivatives were analyzed based on interactions with vital residues and compared the interactions with reference molecules. AGP-16 and 11 showed more stable H-bonds, hydrophobic, and ionic interactions as compared to reported anti-virals. Our present study results are supported by the study reported by Enmozhi et al. (2020) that the amino acid residues involved in the H-bonds interaction of AGP with Mpro are Gly143, Cys145, and Glu166 [12]. The other studies reported by Kodchakorn et al. (2020) [13] and Lashmi et al. (2020) [14] also supporting our present study result as they had reported that natural AGP analogues interacted with His41, Leu141, Asn142, Gly143, Cys154, Met165, Glu166, Gln189, and Met49.
Once the derivative bound to the active site of Mpro, access to this enzyme, which serves as a critical role in the formation of non-structural proteins and the biosynthesis of the virus, is blocked, which leads to no maturation of other nsp and no biosynthesis of the virus. Stable interaction of all the AGP derivatives with key residues and lowest binding energies indicates the potential for these derivatives to inhibit the activity of Mpro and reduce or prevent viral replication.
PLPro, also known as nsp3, is responsible for viral replication and the host innate immunity [34]. In-depth The present study revealed that the most prominent amino acids involved in interaction with the ligand were Tyr264, Pro248, Tyr268, and Tyr273. Identified AGP derivatives AGP14 and 2 in the present study showed more stable H-bonds, hydrophobic, and ionic interactions than reported antivirals. Once the derivative bound to the active site of PLpro, access to this enzyme, which serves as a critical role in replicating the virus, is blocked. The inhibition of PLPro activity by binding to the PLPro catalytic triad can prevent the replication of SARS-CoV-2 and destroy the role of PLPro in the host immune response evasion to reduce the inflammation of host cells [34]. Stable interaction of all the AGP derivatives with key residues and lowest binding energy indicates the potential for these derivatives to inhibit the activity of PLpro and reduce or prevent viral replication. Murugan et al. (2020) reported that the main amino acids involved in the interaction of natural AGP analogues with PLpro areTyr269, Gln270, Gly272, and Tyr274, which support our present study results [16].
SARS-CoV-2 RdRp or non-structural protein 12 (nsp12) is a vital central polymerase involved in RNA replication. It has been studied in various viruses. It is a promising target as studies found that targeting RdRp would not lead to severe toxicity [35].  reported that the active site of RdRp contain-sTyr618, Cys622, Asn691, Asn695, Met755, Ile756, Leu757, Leu758, Ser759, Asp760, Asp761, Ala762, Val763, Glu811, Phe812, Cys813, and Ser814 residue [5]. The active site key residues are adjacent aspartates, i.e., Asp760 and Asp761 [5,36]. Other than REM and AGP, AGP-13 and 6 were interacted with the vital residue Asp760 or Asp761, along with the good binding score. But AGP-14 had better binding score than all other AGP derivatives. The cyclic hydroxyl and carbonyl group of AGP was responsible for the H-bond interaction with RdRp; however, the cyclic carbonyl, nitro, and ester groups of AGP-14 were involved in the five H-bond interactions with RdRp. The other common amino acid residues of the active site involved in the AGP interaction with RdRp were Tyr619, Trp617, Lys621, Ser795, Glu811, and Ser814 (Table S1). Identified AGP derivatives AGP-13, 14, and 6 in the present study showed more stable H-bonds, hydrophobic and ionic interactions than reported antivirals.
For a potent RdRp inhibitor, the derivative or compound must interact with key residues in the active site of the RdRp. No new viral genomes are synthesized when the virus's access to positive sense RNA is blocked by the drug that binds to the RdRP active site. All the identified AGP derivatives, except AGP-3, 4, 5, and 17, have shown stable interaction with key residues, along with the lowest binding energies, which shows the ability of these drugs in inhibition of RdRp, and playing a role in reducing or stopping viral proliferation. Our study results differ from Murugan et al. (2020) as the interaction amino acids with natural AGP analogues reported in their study were Ser644, Arg438, Asp337, Arg509, and Asp508 [16].
Endoribonuclease NSP15 plays a vital role in the replication and life cycle of the virus. The endoribonuclease activity of NSP15 interferes with the innate immune response of the host. All the AGP derivatives and reference compounds have interacted with some of the active site amino acid residues of NSP15: His-235, Asp-240, Ser-242, His-243, Gln-245, Leu-246, Gly-248, His-250, Asn-278, Lys-290, Glu-340, Thr-343, Lys345, and Leu-346 as suggested by Vijayan and Gourinath (2021) [37]. NSP15 is a known anti-viral drug target, and its active site constitutes the catalytic triad His-235, His-250, and Lys-290. All the AGP derivatives except AGP-8, 9, and 13 have interacted with either one or two of the catalytic triad of NSP15. The other essential amino acid residues involved in the active site interaction are Thr-341, Tyr-343, and Ser-294. Identified AGP derivatives AGP-15 and 3 in the present study showed more stable H-bonds, hydrophobic, and ionic interactions than reported anti-viral. Surprisingly the -CH 2 OH group of AGP was involved in three H-bonds formation with NSP15, while the ester group of AGP-15 was responsible for the H-bond interaction with residues of NSP15. However, the methyl substituent at aromatic ring and a cyclic group of AGP-15 were involved in the hydrophobic interaction with NSP15 active site amino acid residues. AGP derivatives binding to the NSP15 active site may potentially block endoribonuclease activity responsible for the protein interference with the innate immune response. These interactions are essential to abolishing viral function and thus reducing the further transmission of the virus. The high binding affinity of AGP derivatives indicates that they can be promising lead compounds as NSP15 inhibitors.
SARS-CoV-2 utilizes the spike protein (S) present on the viral surface to enter the host cells. The protein-protein interaction between the subunits of the spike protein and the ACE-2 receptor active site can be targeted to identify an effective treatment strategy [38]. When spike glycoprotein is inhibited, the ability of the virus to attach to the host cell can be inhibited as well. Identified AGP derivatives AGP-13 and 7 in the present study showed more stable H-bonds and hydrophobic compared to reported antivirals. The ester group of AGP was involved in an H-bond formation and the cyclic ring was responsible for the two hydrophobic interaction with S. However, the aromatic hydroxyl groups of AGP-13 were involved in the two H-bond interactions, and the aromatic ring was responsible for the hydrophobic interaction with S. When AGP derivatives bind to the S active site, spike glycoprotein is inhibited, and the virus's ability to attach to the host cell can also be inhibited. These interactions are essential to abolishing viral function and thus reducing the further transmission of the virus. The high binding affinity of AGP derivatives indicates that they can be promising lead compounds as S inhibitors. Our current study results differ from Lakshmi et al. (2020) [14] and Maurya et al. (2020) [15], since they reported that the natural AGP analogues interacted with Tyr28, Phe59, and Thr761 of spike glycoprotein.

Conclusion
The absence of a most suitable and active drug for curing COVID-19 has already worsened the pandemic condition. The rapidly changing mechanism and mutation of SARS-CoV-2 made us to think and carry out molecular docking studies of andrographolide derivatives with various essential viral proteins/enzymes to reduce or cure viral infection. Interestingly AGP-12, 14, and 15 showed better binding affinities towards the entire tested enzyme than AGP, REM, and HCQ. AGP derivative 16 had shown − 8.7 kcal/mol binding/docking score for Mpro, AGP-15 showed − 8.6 kcal/mol for NSP15, and AGP-10, 13, and 15 exhibited − 8.7, − 8.9, and − 8.7 kcal/mol, respectively, for S. Overall results indicated that AGP derivatives 14 and 15 could be the best 'lead' candidate for the treatment against SARS-CoV-2 infection. However, molecular dynamic studies are essential to confirm the stability of the AGP derivatives 14 and 15 with the SARS-CoV-2 protein complex. The information generated here may provide some insights into exploring potential drugs against SARS-CoV-2 from andrographolide, which can result in the discovery of novel drugs. However, the present study needs further experimental confirmation via in vitro and in vivo studies.